Mycobacterium bovis is a member of the M. tuberculosis complex and the causative agent of tuberculosis (TB) in cattle and zoonotic infections in humans. Bovine TB is an excellent model for understanding TB in humans, as the diseases are parallel in many aspects of pathogenesis and innate and adaptive immune responses. Further, the study of bovine and human TB exemplifies the One Health approach as discoveries in both species have been closely intertwined throughout history. ?? T cells are a set of non-conventional CD3+ T cells that share important characteristics of both the innate and adaptive arms of the immune system. ?? T cells are particularly recognized for their ability to respond robustly to Mycobacterium infection. Several characteristics of ?? T cells, predominantly those functions commonly shared with ?? T cells, such as IFN? production have been identified and characterized; however, much less is known about the alternative immune functions of ?? T cells such as chemokine production and regulatory cytokine production. We have previously prepared samples of highly purified ?? T cells from animals infected with virulent M. bovis and in Aim 1, we propose to employ next-generation RNA-Sequencing on these samples to identify novel and alternative functions for ?? T cells responding to Mycobacterium. ?? T cells are hypothesized to play a critical role in granuloma formation and immune cell recruitment; however, given the difficulty of studying the immune response to TB in vivo, little is known about the functions of ?? T cells at the site of infection. Therefore, in Aim 2 we propose to utilize RNAScope, a commercial technology similar to in situ hybridization to analyze the cytokines and chemokines produced by ?? T cells present at the site of M. bovis granulomas. If successful, the studies from our RNA-Seq analysis will further inform our studies of ?? T cell functions in infected tissues. Ours will be one of the first studies to correlate ?? T cell responses in the peripheral blood, with those in the tissues during M. bovis infection. The knowledge gained from our studies will contribute to our understanding of the basic biology of ?? T cells and are anticipated to significantly advance the fields of human and veterinary medicine alike.